title: ER stress activating ATF4/CHOP-TNF-α signaling pathway contributes to alcohol-induced disruption of osteogenic lineage of multipotential mesenchymal stem cell.
abstract: OBJECTIVE
  Studies have provided substantial evidence that osteoblasts and adipocytes share common progenitor‒multipotential mesenchymal stem cells in bone marrow (BMSCs), and excessive alcohol consumption shifts away from osteogenic to adipogenic lineage. However, how exactly alcohol impairs osteogenesis is still incompletely understood. This study was designed to shed light on this issue.
  
  
  METHODS
  We treated primary BMSCs from human subjects with alcohol for 24 days. We measured changes of genes related to endoplasmic reticulum (ER) stress, adipogenic markers and osteogenic markers using quantitative real-time RT-PCR and Western blot analysis. We performed Oil red O staining and quantification of adipogenesis. We also conducted caspase 3 activity assay to assess BMSC apoptosis.
  
  
  RESULTS
  We showed here that chronic exposure of BMSCs to alcohol induced adipogenesis and disrupted osteogenesis as indicated by upregulation of adipogenic markers (PPARγ2 and aP2), downregulation of osteogenic markers (Osf2/Cbfa1), and accumulation of lipid droplets. Alcohol induced ER stress, as reflected by increased expression of glucose-regulated proteins GRP78 and GRP94, and by increased expression of transcription factors activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and enhanced caspase 3 activity. Additionally, ER stress also upregulated tumor necrosis factor-alpha (TNF-α). Simultaneous silencing of ATF4 and CHOP prevented upregulation of TNF-α. Knockdown of either ATF4 and CHOP or TNF-α by their siRNAs was able to reverse the ethanol-induced adipogenesis.
  
  
  CONCLUSIONS
  Our data therefore revealed a role of ER stress and ATF4/CHOP in the ethanol-induced inhibition of osteogenesis, and activation of TNF-α signaling by ATF4/CHOP linking ER stress to adipogenic lineage in response to alcohol stimulation. This work should establish a new signaling pathway linking alcohol, ER stress, and TNF-α to loss of bone formation: Ethanol → ER stress↑↑↑ → ATF4 & CHOP↑↑↑ → TNF-α↑↑↑ → Osteoblasts↓↓↓.
authors.affiliation: Southern Medical University, 2010 MD TongHe, Guangzhou, China.
authors.forename: Yueping
authors.initials: Y
authors.lastname: Chen
authors.forename: Hui
authors.initials: H
authors.lastname: Gao
authors.forename: Qingshui
authors.initials: Q
authors.lastname: Yin
authors.forename: Liang
authors.initials: L
authors.lastname: Chen
authors.forename: Panfeng
authors.initials: P
authors.lastname: Dong
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Zhang
authors.forename: Jie
authors.initials: J
authors.lastname: Kang
mesh_terms: D051701:Activating Transcription Factor 4 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050156:Adipogenesis / Q000187:drug effects; D001854:Bone Marrow Cells / Q000166:cytology; D053148:Caspase 3 / Q000378:metabolism; D019070:Cell Lineage / Q000187:drug effects*; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D000431:Ethanol / Q000633:toxicity*; D050556:Fatty Acid-Binding Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects; D047495:PPAR gamma / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051742:Transcription Factor CHOP / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D000091342:Endoplasmic Reticulum Chaperone BiP; C493410:FABP4 protein, human; D050556:Fatty Acid-Binding Proteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D008562:Membrane Glycoproteins; D047495:PPAR gamma; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C053387:endoplasmin; D051701:Activating Transcription Factor 4; D051742:Transcription Factor CHOP; D000431:Ethanol; D053148:Caspase 3